The present invention relates to monoclonal antibodies produced by hybrid cell lines characterized in that the antibodies have specificity to human colon fibroblast-derived tissue plasminogen activator (t-PA). The present invention also relates to the use of the monoclonal antibodies in a method for the purification of t-PA and in a method for the immunoassay of t-PA.
With the advent of hybridoma technology first developed by Kohler and Milstein, it is now possible to generate monoclonal antiboides which are essentially homogenous compositions having uniform affinity for a binding site. The production of mouse hybridomas by these investigators is described in Nature 256, 495-497 (1975); and Eur. J. Immunol. 6, 511-519 (1976). According to this method, tissue-culture adapted mouse myeloma cells are fused to spleen cells from immunized mice to obtain the hybrid cells that produce large amounts of a single antibody molecule. The fusion is generally carried out in the presence of polyethylene glycol (PEG) as described by Galfe et al., Nature 266, 550-552 (1977), followed by selection in HAT medium (hypoxanthine, aminopterin and thymidine) as described by Littlefield, Science 145, 709-710 (1964).
While immunization can be carried out with virtually any foreign antigen of interest, many difficulties arise and variations are required for each specific case. Prior to attempting to prepare a given hybridoma, there is no assurance that the desired hybridoma will be obtained, that it will produce antibody if obtained, or that the antibody so produced will have the desired specificity.
A number of publications have described the preparation of hybridomas that produce monoclonal antibodies against t-PA derived from Bowes melanoma cultured cells, human plasma and human uterine tissue. See, for example, Pettersson et al., Haemostasis 11(Supp. 1), p. 75, abstract 134 (1982); Pettersson et al., Prog. Fibrinolysis 6, 191-194 (1983); Nielsen et al., The EMBO J., 2(1), 115-119 (1983); Matsuo et al., Thromb. Res. 36, 517-526 (1984); MacGregor et al., Thromb. Haemos. 53(1), 45-50 (1985); Schleef et al., Ibid., 53(1), 170-175 (1985); Angles-Cano, Blood 66(4), 913-920(1985); Holvoet et al., Blood 67(5), 1482-1487(1986); and UK patent application No. GB 2,122,219, published Jan. 11, 1984. Such hybridomas have been used to produce monoclonal antibodies which have been used for in vitro purification of Bowes melanoma t-PA as described, for example, by Einarsson et al., Biochim. Biophys. Acta 830, 1-10 (1985); and Reagan et al., Thromb. Res. 40, 1-9 (1985). Several of these monoclonal antibodies against Bowes t-PA are available commercially, e.g. from American Diagnostica Incorporated, Greenwich, Conn. (ADI).
Recently, in copending application Ser. No. 849,933, filed Apr. 9, 1986, three of the present inventors together with others described a process for preparing human colon fibroblast-derived t-PA. The unique, heterogeneous glycosylation pattern in this t-PA is described in copending application Ser. No. 834,080, filed Feb. 26, 1986, by one of the present inventors together with others now U.S. Pat. No. 4,751,084. The disclosures of said copending applications which are assigned to a common assignee, are incorporated by reference herein.
Monoclonal antibodies against human colon fibroblast t-PA have not been described heretofore although it has been reported by Tissot and Bachman, Prog. Fibrinolysis 6, 133-135 (1983), that monoclonal antibodies against Bowes melanoma t-PA recognize t-PA from colon tissue.